Earlier, we generated a conditional transgenic mouse line in which diphtheria toxin receptor was selectively expressed in mossy cells using the Cre/loxP system. Within one week after diphtheria toxin injection, we observed 80% loss of mossy cells throughout the longitudinal axis. We found no obvious or sustained epilepsy-like discharges in the hippocampus as measured by in vivo local field potential recordings. Interestingly, no mossy fiber sprouting was detected by Timm staining. These results suggested that, in contrast to previous reports showing that lesions of the entire hilar region induce massive mossy fiber sprouting and epilepsy, selective in vivo elimination of mossy cells does not trigger behavioral epilepsy or mossy fiber sprouting. This year, we found that dentate granule cells in the DT-treated mutants became hyperexcitable to afferent stimulation in in vitro slice preparation, and during this hyperexcitable state deficits in contextual pattern separation were detected. We also evaluated the immediate-early gene (IEG) expression in response to kainic acid (KA) injection under the assumption that an excitatory stimulus would cause more granule cells to discharge and activate IEG expression in mutants compared to controls. KA injection evoked Zif268 expression in more granule cells in mutants than in controls. We also examined the KA-induced seizure intensity. The cumulative seizure score of mutants for the hour following KA injection was significantly higher than controls. Together, these results all suggested an increase in granule cell excitability following mossy cell ablation. In summary, we concluded that mossy cell loss in vivo renders the granule cells hyperexcitable. Contrary to the predicted epileptogenesis implicit in the dormant basket cell hypothesis, however, it was insufficient to trigger the mossy fiber sprouting and epileptic discharges. Perhaps, in addition to the loss of mossy cells, neurodegeneration of other limbic areas, such as entorhinal cortex, is necessary to induce medial temporal lobe epilepsy. These findings provide new insights into the mechanisms of epileptogenesis in the limbic cortex. This year we conducted several additional experiments for the mossy cell ablation project, which were suggested by reviewers during the manuscript revision process. First, we needed to have decisive evidence for a major feed-forward inhibitory projection via local interneurons from mossy cells to dentate granule cells. Second, we needed to uncover the time course of mossy cell ablation in the hilus. To determine the nature of the mossy cell projections to the granule cells, we measured spontaneous IPSCs (sIPSCs) in the presence of glutamate receptor blockers (AP5 and NBQX) in hippocampal slice preparation. We had already demonstrated prior to this year that, in the absence of those blockers, the frequency of sIPSCs from dentate granule cells were reduced in the mutants compared to the control mice 5-7 days after DT treatment. This suggested that the inhibitory inputs to granule cells are reduced upon mossy cell ablation. If this reduction disappears in the presence of glutamatergic excitatory receptor blockers in the mutant slices, it would indicate the presence of a feed-forward inhibitory projection from mossy cells to granule cells via local interneurons. In other words, glutamatergic mossy cell projections excite local inhibitory interneurons, which in turn inhibit mossy cells. Indeed, we confirmed no difference in the sIPSC frequency in the presence of the blockers between genotypes, suggesting a feed-forward inhibition from mossy cells to granule cells. To uncover the time course of mossy cell ablation in the hilus we used the dye Fluoro-Jade B (FJB), which stains the neurodegeneration during the process of the cell ablation. We found that by post-treatment day 3-4, mossy cell somata were FJB-stained, and these were gone by post-treatment day 7, which was confirmed by anti-calretinin staining, a marker of ventral mossy cells. Presently the manuscript is still under revision while we combine this data with other immunocytochemistry still under way.